The art of delivery systems OZ Biosciences / Protocol Mag4C-Lv Kit / / - 0 -

Transcription

1 The art of delivery systems OZ Biosciences / Protocol Mag4C-Lv Kit / / - 0 -

2 Mag4C-Lv Kit Instruction Manual Mag4C-Lv Kit for magnetic capture, and conservation of Lentiviruses and Retroviruses List of Mag4C-Lv Kits Catalog Number Description Contents Number of Captures* LTK11200 Mag4C-Lv Trial kit Mag4C-Lv beads (0.2 ml) Up to 20 Elution Buffer (5 ml) Conservation Buffer (0.2 ml) LKC11000 Mag4C-Lv kit Mag4C-Lv beads (1mL) Up to 100 Elution Buffer (5mL) Conservation Buffer (1mL) MSR1000 Magnetic Separation rack Magnetic Separation rack NA * Number of captures based on 1 ml of virus preparation. Use the content of the table above to determine the appropriate catalog number for your needs. You can order these products by contacting us. For all other supplementary information, do not hesitate to contact our dedicated technical support (tech@ozbiosciences.com). The art of delivery systems OZ Biosciences / Protocol Mag4C-Lv Kit / / - 1 -

3 Table of Contents 1. Technology Description Kit Contents 3 2. Applications Virus Types Downstream Biological Assays 3 3. Magnetic separation rack and Magnetofection Apparatus 3 4. Protocol General Considerations Capture/Concentration Protocol Elution Procedure Conservation Procedure Magnetofection Procedure Appendix Critical Parameters for best performance Optimization Troubleshooting Quality Controls Related Products Purchaser Notification Technology 1.1. Description Mag4C-Lv Kit is specifically designed and developed for capturing, concentrating and storing Lentiviruses and Retroviruses. This kit is composed of 3 reagents allowing Magnetic Capture/Concentration, Elution and Conservation of Lentiviruses/Retroviruses and a multipurpose Magnetic Separation Rack. Mag4C-Lv magnetic nanoparticles capture by electrostatic and hydrophobic interactions viruses in culture medium with % efficiency. Once captured onto magnetic beads, viruses can be: (1) Concentrated and stored with the Conversation Buffer or directly used for cell culture, molecular biology or other assays. (2) Concentrated, eluted from the magnetic beads with the Elution Buffer and stored with the Conversation Buffer or used for various assays. The Conservation Buffer has been expressly designed to improve the stability of Lentiviruses/Retroviruses upon storage conditions. This buffer is fully compatible with magnetic nanoparticles, meaning that virus bound to magnetic beads can be diluted directly into the buffer for long term storage. Mag4C-Lv Kit is dedicated to Lentiviruses/Retroviruses and presents unique properties: For 1. Concentration of viruses by magnetic capture in minutes 2. Simple, rapid & ready-to-use: No need to process magnetic beads before capture 3. High yield of viral capture and recovery 4. Fast (2 to 1000 X) 5. Avoid ultracentrifugation, precipitation and chemicals: no stringent buffer or physical action on viruses 6. Reduced handling steps of viruses (minimized bio-hazard) 7. Suitable for large volumes 8. Serum compatible & Non Toxic 9. Ideal for cell culture transduction/infection (Magnetofection advantages) For conservation 1. Improved virus preservation upon storage (-80 C) 2. Maintain high virus titers upon freeze and thaw cycle 3. Compatible with magnetic nanoparticles OZ Biosciences / Protocol Mag4C-Lv Kit / / - 2 -

4 1.2. Kit Contents OZ Biosciences offers two sizes of Mag4C-Lv Kit (trial and kit). Mag4C-Lv Kit contents: 1 tube containing 0.2 ml (trial) or 1 ml of Mag4C-Lv beads 1 tube containing 5 ml of Elution Buffer (1X). 1 tube containing 0.2 ml (trial) or 1 ml of Conservation Buffer (5X). The kit do not contain the Magnetic Separation Rack that must be purchased separately Stability and Storage Mag4C-Lv beads, Elution Buffer Storage and Conservation Buffer Storage: +4ºC. Magnetic Separation Rack can be stored at room temperature Kit components are stable for at least one year at the recommended storage temperature. Do not freeze the Mag4C-Lv beads! Do not add anything to the Mag4C-Lv beads and buffers! Shipping condition: Room Temperature 2. Applications 2.1. Virus Types Mag4C-Lv beads can be combined with any Lentiviruses/Retroviruses. An updated list of viruses successfully tested is available on the website: Downstream Biological Assays After magnetic capture and, viruses can be used for multiple assays. For instance, they can be used for PCR, western blot, ELISA, in vitro and in vivo infection, etc. Viruses can be directly used with the bound Mag4C- Lv beads or eluted from the Mag4C-Lv beads (free of beads). For cell biology, we suggest to use viruses associated with Mag4C-Lv beads to infect cells. Virus complexed to Mag4C-Lv beads, eluted virus (free of beads), and virus in conservation buffer have all been successfully tested on a variety of immortalized and primary cells. Please consult our list of cells successfully tested available on the website: For in vitro and in vivo infection. Mag4C-Lv beads are compatible with the Magnetofection technology. This method allows concentrating the entire viral dose on the cells very rapidly, accelerating the transduction process and infecting non-permissive cells. Moreover, virus infection efficiency is significantly increased and cell adsorption/infection can be synchronized without modification of the viruses. Targeted/confined transduction to specific area (magnetic targeting) can also be accomplished. For more information visit 3. Magnetic separation rack and Magnetofection apparatus Mag4C-Lv Kit requires appropriate magnetic fields for concentrating magnetized viruses. The Magnetic Separation Rack (#MSR1000, photo), is designed for 50, 15 or 1.5 ml tubes. It can hold 12 standard microtubes, two 15 ml and two 50 ml tubes. The Magnetic Separation Rack is required for capture,, washing and elution. If Mag4C-Lv beads are kept bound to viruses and to take advantage of the Magnetofection technology for the downstream cell culture or in vivo assays, a magnetic plate or magnets are needed. OZ Biosciences provide specific magnetic plates for Magnetofection : 96-magnets plate (#MF10096), super magnetic plate (#MF10000) and mega magnetic plate (#MF14000) and specific magnets set for in vivo applications (#IV-MAG1). OZ Biosciences / Protocol Mag4C-Lv Kit / / - 3 -

5 4. Protocol 4.1. General Considerations The instructions given below represent typical protocols that were applied successfully to Capture, Concentrate, Elute, and Conserve freshly produced or purchased viruses. Our R&D team has tested and optimized the Mag4C-Lv Kit in order to provide you with the most straightforward and efficient procedure. Therefore, we suggest you to start by following our general protocol as guidelines to obtain good data rapidly. Thereafter, we recommend optimizing the conditions to achieve the best performance. Indeed, optimal conditions vary from one virus production to another and are highly dependent upon the type of virus used, its titer, the composition of the viral solution, and cell culture conditions. We advise you to optimize the experimental condition parameters as described in the Appendix in order to achieve the best effects Capture/Concentration Protocol The protocol is simple: 20 µl of Mag4C-Lv beads are sufficient to bind 1x10 6 infectious viruses with almost 80-99% efficiency. Please refer to the Table 1 for the suggested Mag4C-Lv beads volume according to the virus titer. Depending on the virus type, the total virus quantity (particles and infectious), and the complexity of the medium, this protocol would have to be adjusted (see appendix). It is recommended to raise the volume of Mag4C-Lv beads for complex medium (complete culture medium, organic fluids ). Table 1 Recommended volume of Mag4C-Lv beads according to the number of infectious viruses Viral Preparation (ml) Mag4C-Lv beads ( L) Starting Point * Mag4C-Lv beads (µl) Suggested range of testing 2 ml 20 µl 10 µl 40 µl > 2mL 10 µl / ml 5 µl 20 µl / ml * for high titer viral solution ( 10 7 infectious viruses / ml), we recommend using 1.5 or 2 times the suggested volume. Important: The suggested volume of Mag4C-Lv beads for capture is related to infectious particles and not physical viral particles. 1) Add 20 µl of Mag4C-Lv beads into the virus preparation 2 ml or 10 µl / ml of Mag4C-Lv beads for virus preparation > 2 ml. It may be necessary to adjust the volume of Mag4C-Lv depending on the composition of the virus solution (see Table 1). 2) Incubate min at room temperature to capture viruses. 3) Place the tube 15 to 30 min onto the Magnetic Separation Rack to concentrate the virus/mag4c-lv beads complexes. Incubation time will depend on the tube volume (see Table 2). Then, discard supernatant. NOTE: Brown pellet should be visible on the side of the tube near the magnets. OZ Biosciences / Protocol Mag4C-Lv Kit / / - 4 -

6 OPTIONAL: A washing procedure can be performed after this step: i. Keep the tube on the Magnetic Separation Rack and slowly add PBS (same volume as the initial medium). ii. Incubate 5 min on the Magnetic Separation Rack. iii. Discard the supernatant. iv. Proceed to step 4 Table 2 Recommended incubation time on the Magnetic Separation Rack according to the volume Tube volume Time on Magnetic Separation Rack 1 ml 15 min 10 ml 20 min 50 ml 30 min 4) The virus/mag4c-lv beads complexes can be used according to the following 4 options (1) viruses are concentrated with Mag4C-Lv beads into smaller volumes of PBS (with Ca 2+ and Mg 2+ ) or complete cell culture medium and used immediately for assay (for example see 4.5). Determine the appropriate volume of PBS/medium to add according to the expected final. (2) viruses are concentrated with Mag4C-Lv beads into smaller volumes of Conservation Buffer for long term storage (see 4.4) (3) viruses are eluted from Mag4C-Lv beads (see 4.3), concentrated into smaller volumes of Elution Buffer and used immediately for assay (4) viruses are eluted from Mag4C-Lv beads (see 4.3), concentrated into smaller volumes of Elution Buffer plus Conservation Buffer for long term storage (see 4.4) Keeping the Mag4C-Lv beads bound to viruses offers several advantages especially in terms on in vitro and in vivo infectivity as it allows using the Magnetofection technology (see 4.5): OZ Biosciences / Protocol Mag4C-Lv Kit / / - 5 -

7 4.3. Elution Procedure This step is optional. You can choose to keep the nanobeads associated to the virus or remove them and have a beads-free concentrated virus. The Elution Buffer is a ready-to-use buffer, specifically designed to elute lentiviruses bound to the Mag4C-Lv nanobeads without impairing their infectious properties. Important Note: For storage of eluted viruses, go directly to the section 4.4 1) Proceed to capture the virus as previously described and discard the supernatant (see 4.2 steps 1 to 3). Add the Elution Buffer to the virus/mag4c-lv beads complexes. Determine the appropriate volume of Elution Buffer to add according to the expected final (see Table 3). For example, if the initial virus solution is 1mL and you want to concentrate 10 fold, then add 100µL of Elution Buffer. If you just want to wash or exchange the medium of the virus solution (no ), add the same volume of Elution Buffer as the initial virus solution (see Table 3). Table 3 Volume of Elution Buffer for and immediate use Starting viral solution Expected 100X Expected 50X Expected 10X Washing or medium exchange 1 ml 10 µl 20 µl 100 µl 1 ml 5 ml 50 µl 100 µl 500 µl 5 ml 10 ml 100 µl 200 µl 1 ml 10 ml 50 ml 500 µl 1 ml 5 ml 50 ml 2) After addition of the Elution Buffer, incubate for 5 to 10 min at RT. 3) Place the tube on the Magnetic Separation Rack and incubate 10 to 30 min at RT. NOTE: Adjust incubation time on the Magnetic Separation Rack according to the volume (refer to table 2). 4) Save the supernatant containing viruses and discard pellet of Mag4C-Lv nanobeads. 5) The concentrated viruses solution can be used for downstream assay or proceed to section 4.4 for storage Conservation Procedure Conservation procedure with Mag4c-Lv nanobeads. Conservation buffer is 100% compatible with Mag4C-Lv nanobeads. In this way, Conservation Buffer can be added right after the capture procedure. Conservation Buffer allows virus storage for several months at -80 C and preservation of the virus titer upon freeze/thaw cycles. OZ Biosciences / Protocol Mag4C-Lv Kit / / - 6 -

8 Conservation Buffer preparation: Dilute 1 volume of the Conservation Buffer (5x) in 4 volumes of PBS (1X final). For 100 µl conservation buffer, add 20 µl of buffer to 80 µl of PBS. 1) Proceed to capture the virus as previously described and discard the supernatants (see 4.2 steps 1 to 3) 2) Remove the tube form the Magnetic Separation Device 3) Add freshly prepared conservation buffer to the complexes. To concentrate the virus solution, use smaller volume of buffer. 4) Store the complexes at -80 C. NOTE: To reduce freezing/thawing cycles, it is recommended to aliquot virus for long term storage. Conservation procedure after elution. Conservation Buffer can be added right after the elution step. Conservation Buffer allows virus storage for several months at -80 C and preservation of the virus titer upon freeze/thaw cycles. 1) Proceed to capture the virus as previously described and discard the supernatants (see 4.2 steps 1 to 3) 2) Add the Elution Buffer to the virus/mag4c-lv beads complexes (see Table 4 for Elution Buffer volume). Table 4 Volume of Elution Buffer (EB) and Conservation Buffer (CB) for and storage Starting viral solution Expected 100X Expected 50X Expected 10X Washing or medium exchange EB CB EB CB EB CB EB CB 1 ml 8 µl 2 µl 16 µl 4 µl 80 µl 20 µl 800 µl 200 µl 5 ml 40 µl 10 µl 80 µl 20 µl 400 µl 100 µl 4 ml 1 ml 10 ml 80 µl 20 µl 160 µl 40 µl 800 µl 200 µl 8 ml 2 ml 50 ml 400 µl 100 µl 800 µl 200 µl 4 ml 1 ml 40 ml 10 ml 3) After addition of the Elution Buffer, incubate for 5 to 10 min at RT. 4) Place the tube on the Magnetic Separation Rack and incubate 10 to 30 min at RT. NOTE: Adjust incubation time on the Magnetic Separation Rack according to the volume (refer to table 2). 5) Save the supernatant containing viruses and discard pellet of Mag4C-Lv nanobeads. 6) Add the Conservation Buffer (5x) directly to the eluted viruses solution for a 1X final. See Table 4. 7) Store virus at -80 C NOTE: To reduce freezing/thawing cycles, it is recommended to aliquot virus for long term storage. OZ Biosciences / Protocol Mag4C-Lv Kit / / - 7 -

9 4.5. Magnetofection Procedure Keeping the Mag4C-Lv beads bound to viruses offers the Magnetofection technology advantages. This method allows concentrating the entire viral dose on the cells quickly, accelerating the transduction process and infecting non-permissive cells. The virus infection efficiency is considerably increased and virus adsorption can be synchronized without modification of the virus. Targeted/confined transduction to specific area (magnetic targeting) can also be accomplished. You can find more information at or in the Mag4C-Kit results. After capture, and storage (with the nanobeads), the Magnetofection procedure can be performed for in vitro transduction or in vivo infection. Specific protocols are available directly on our website for in vitro and in vivo experiments. Magnetofection in vitro on adherent cells. 1) Perform the capture and of the virus as described above (see 4.2, steps 1-4) or thaw the virus/mag4c-lv beads complexes vial (4.4 conservation with the beads). 2) This protocol is given for a 24-well plate format; refer to the Magnetofection protocol for other sizes of culture dishes. 3) Plate the cells the day prior transduction. Best results are achieved if cells are at least % confluent at the time of Magnetofection (if required refer to the suggested cell number in the Magnetofection protocol). 4) Add the virus/mag4c-lv beads complexes to the cells at the desired MOI in a drop wise manner. OZ Biosciences / Protocol Mag4C-Lv Kit / / - 8 -

10 5) Mix by gently rocking the plate to ensure correct dispersion of magnetic complexes within culture medium. 6) Place the cells upon the specific magnetic plate (see section 3) for minutes. NOTE: Optionally after this incubation, a medium change can be performed while maintaining the magnetic plate under the cell culture. 7) Remove the magnetic plate and cultivate the cells under standard conditions until evaluation of the transduction experiment. NOTE: Optionally a medium change can be performed after 24 hours. For suspension cells, please refer to the Magnetofection protocol (ViroMag and ViroMag R/L or Viro-MICST) For in vivo, please refer to the in vivo Magnetofection protocol For more information on Magnetofection procedures contact: tech@ozbiosciences.com 5. Appendix 5.1. Critical Parameters for best performance 1) Mag4C-Lv quantity. We usually observe good capture efficiency with 20µL of Mag4C-Lv beads for 10 6 infectious viral particles. However the efficiency may depend on the virus type used and the medium composition. Consequently, we suggest you to start by testing a range of Mag4C-Lv volumes in order to obtain the best experimental conditions. 2) Magnetic Separation. The capture, and elution on the Magnetic Separation Rack depend on the amount/ of virus used. Indeed, longer incubation under the magnetic field might be required with very low viral titers whereas with high viral dose short incubation times are sufficient. 3) Elution Buffer. The volume of Elution Buffer as well as the incubation time for the elution might have to be adjusted depending on the virus and the amount of Mag4C-Lv beads. 4) Conservation Buffer. Conservation buffer preserves lentivirus effect after -80 C storage with multiple freezing/thawing cycles. However, we recommend to aliquot viral particles solution in conservation buffer to avoid virus degradation and thus ensuring the best conservation conditions Optimization In order to get the best out of Mag4C-Lv Kit, several parameters can be optimized. OZ Biosciences team has investigated numerous factors during the course of the R&D program. Based on our experience, we recommend you to start from the experimental procedures described above (section 4) and optimize one parameter at a time 1) Start by optimizing the Mag4C-Lv beads dose. To this end, vary the amount of Mag4C-Lv in the range suggested in the Table 1. For instance, for 10 6 viral infectious particles or for viral preparation < 2mL use 10 to 40 µl of Mag4C-Lv. After the capture step, you can test the supernatant for remaining infectivity. If capture is efficient, the supernatant should not be infectious. 2) Depending on the quantity of virus the time of incubation for the complexes formation as well as for the capture on the Magnetic Separation Rack should be extended. 3) If using a complex medium (organic fluid ), we recommend diluting sample with PBS at least 1.5 times. Do not dilute medium excessively to avoid large dispersion of viral particles that hamper efficient virus capture. OZ Biosciences / Protocol Mag4C-Lv Kit / / - 9 -

11 5.3. Troubleshooting Our dedicated and specialized technical support team will be pleased to answer any of your requests and to help you with your experiments at In addition, do not hesitate to visit our website for more information or to contact us at Problems Comments and Suggestions Low capture and 1. Virus titer. Ensure that virus titer used corresponds to infectious viral particles and not physical particles. A 1 to 1000 ratio can be found between physical and efficiency. infectious particles. 2. Virus quality. Even though Mag4C-Lv has been designed to work with culture medium containing viral particles, for the best efficiency viruses should be as pure as possible, exempt of contaminants 3. Medium Composition. Use higher volume of Mag4C-Lv beads to raise the capture efficiency. If too viscous, medium can be diluted with PBS (with Ca 2+ and Mg 2+ ) or with salt balanced physiological buffer at least 1.5 times. 4. Capture time. 15 min should lead to an optimal capture. Still it may be necessary to incubate Mag4C-Lv beads longer with virus to ensure complete capture. 5. Medium purity. Be sure that no contaminants are present in medium. Endotoxins or microorganisms will easily bind on beads and compete with virus fixation. 6. Reagents temperature. Reagents should have an ambient temperature and be vortexed prior to use Low elution 1. Incubation time. 10 min in presence of Elution Buffer and 30 min on separation efficiency rack should lead to optimal elution. It may be necessary to increase both incubation periods to 20 min and 45 min respectively. 2. Temperature. Perform the elution process at 37 C. 3. Washing. Before elution procedure, wash magnetic complexes with PBS as suggested in paragraph 4.2. Low infection 1. Cell density. A non-optimal density can lead to poor efficiency. The optimal efficiency with or confluency should range from 50 to 90% (true confluency, corresponding to 90% without visual confluency) but most favorable density may vary according to the cell type. Magnetofection 2. Type of virus. Ensure that the virus can infect (being expressed) the cells. Another viral-driven promoter can be used as a control. 3. Cell condition. Use freshly thawed cells that have been passaged at least once. Cells should be healthy and assayed during their exponential growing phase. The presence of contaminants (mycoplasma, fungi) can alter the transduction efficiency. 4. Incubation time and transduction volume. 1) The optimal time range between transduction and assay varies with cells, promoter, expression product, etc The transduction efficiency can be monitored after 24 96h by analyzing the gene product. Several reporter genes can be used to quantitatively monitor gene expression kinetics. 2) To increase transduction efficiency, transfection volume can be reduced for the first 24 hours. 5. Transgene detection assay. Ensure that your post-transduction assay is properly set up and includes a positive control. 6. Multiplicity of Infection (MOI). Be sure that the MOI is properly calculated. There could be a 1/10 to 1/1000 ratio between physical and infectious particles. 7. shrna design. The design of an efficient shrna is crucial. Ensure to use a validated shrna sequence encoded in the expression vector. Cellular toxicity 1. Unhealthy cells. 1) Check cells for contamination, 2) Use new batch of cells, 3) Ensure culture medium condition (ph, type of medium used, contamination etc ), 4) Cells are too confluent or cell density is too low, 5) Verify equipments and materials. 2. Infection is toxic. Most of the lentiviruses used are not replicative. Be sure that the cell line doesn t express the missing region for replication. It should be noted that even if lentivirus can t replicate into the cells, it can still express viral proteins that can be toxic and cause cytopathic effect. Oncolytic viruses kill cells. 3. Concentration of Mag4C-Lv/virus complexes too high. Decrease the amount of complexes added to the cells by lowering the MOI. 4. Incubation time. Reduce the incubation time of complexes with the cells by replacing the transfection medium by fresh medium after 4 h to 24 h. 5. Key gene silencing. If the targeted gene is essential for cell survival or if a key gene is non-specifically silenced by the si- or shrna, this can lead to cell death. OZ Biosciences / Protocol Mag4C-Lv Kit / /

12 5.4. Quality Controls To assure the performance of each batch of Mag4C-Lv Kit produced, we qualify each lot using rigorous standards. In vitro assays are conducted to qualify the quality and activity of each kit component. Components Mag4C-Lv & buffers Mag4C-Lv & buffers Magnetic Separation Rack Standard Quality Controls 1. Sterility. Thioglycolate assay: absence of fungal and bacterial contamination shall be obtained for 14 days. 2. Quality and size homogeneity of the magnetic nanoparticles. 3. Stability of the magnetic nanoparticles formulations. 4. Mag4C-Lv capture and transduction efficacies with a recombinant lentivirus on NIH-3T3 or HeLa cells. Every lot shall have an acceptance specification of > 80% of the activity of the reference lot. 5. Elution and preservation performance 6. Tests of solidity and Test of the magnetic field force 6. Related Products Description MAGNETOFECTION TECHNOLOGY Super Magnetic Plate (standard size for all cell culture support) Mega Magnetic plate (mega size to hold 4 culture dishes at one time) Transfection reagents: PolyMag Neo (for all nucleic acids) Magnetofectamine (for all nucleic acids) NeuroMag (dedicated for neurons) SilenceMag (for sirna application) Transfection enhancer: CombiMag (to improve any transfection reagent efficiency) Viral Transduction enhancers: ViroMag R/L (specific for Retrovirus and Lentivirus) AdenoMag (for Adenoviruses) LIPOFECTION TECHNOLOGY (LIPID-BASED) Lullaby (sirna transfection reagent) DreamFect Gold (Transfection reagent for all types of nucleic acids) VeroFect (for Vero cells) FlyFectin (for Insect cells) i-micst TECHNOLOGY Viro-MICST (to transduce directly on magnetic cell purification columns) 3D TRANSFECTION TECHNOLOGY 3Dfect (for scaffolds culture) / 3DfectIN (for hydrogels culture) RECOMBINANT PROTEIN PRODUCTION HYPE-5 Transfection Kit (for High Yield Protein Expression) PROTEIN DELIVERY SYSTEMS Ab-DeliverIN (delivery reagent for antibodies) Pro-DeliverIN (delivery reagent for protein in vivo and in vitro) PLASMIDS PVECTOZ pvectoz-lacz / pvectoz-seap / pvectoz-gfp / pvectoz-luciferase ASSAY KITS Bradford Protein Assay Kit MTT cell proliferation kit β-galactosidase assay kits (CPRG/ONPG) BIOCHEMICALS D-Luciferin, K + and Na + 1g X-Gal powder 1g / G-418, Sulfate 1g Please, feel free to contact us for all complementary information and remember to visit our website ( to stay informed on the latest breakthrough technologies and updated on our complete product list. OZ Biosciences / Protocol Mag4C-Lv Kit / /

13 Purchaser Notification Limited License The purchase of the Mag4C-Lv Kit and other Magnetofection Reagents grants the purchaser a non-transferable, non-exclusive license to use the kit and/or its separate and included components (as listed in section 1, Kit Contents). Reagents are intended for in-house research only by the buyer. Such use is limited to the capture,, elution, conservation and transduction of virus as described in the product manual. In addition, research only use means that this kit and all of its contents are excluded, without limitation, from resale, repackaging, or use for the making or selling of any commercial product or service without the written approval of OZ Biosciences. Separate licenses are available from OZ Biosciences for the express purpose of non-research use or applications of the Mag4C-Lv Kit and other Magnetofection Reagents. To inquire about such licenses, or to obtain authorization to transfer or use the enclosed material, contact the Director of Business Development at OZ Biosciences. Buyers may end this License at any time by returning all Mag4C-Lv Kit and other Magnetofection Reagents material and documentation to OZ Biosciences, or by destroying all Mag4C-Lv Kit and other Magnetofection Reagents components. Purchasers are advised to contact OZ Biosciences with the notification that a Mag4C-Lv Kit and other Magnetofection Reagents kit is being returned in order to be reimbursed and/or to definitely terminate a license for internal research use only granted through the purchase of the kit(s). This document covers entirely the terms of Mag4C-Lv Kit and other Magnetofection Reagents research only license, and does not grant any other express or implied license. The laws of the French Government shall govern the interpretation and enforcement of the terms of this License. Product Use Limitations The Mag4C-Lv Kit and other Magnetofection Reagents and all of its components are developed, designed, intended, and sold for research use only. They are not to be used for human diagnostic or included/used in any drug intended for human use. All care and attention should be exercised in the use of the kit components by following proper research laboratory practices. For more information, or for any comments on the terms and conditions of this License, please contact: Director of Business Development OZ Biosciences Parc Scientifique et Technologique de Luminy 163, avenue de Luminy, zone entreprise, case Marseille Cedex 9, France Tel: +33 (0) Fax: +33 (0) business@ozbiosciences.com OZ Biosciences / Protocol Mag4C-Lv Kit / /